Increasing evidence suggests important events in T cell maturation in the thymus are governed by specific gene regulation. In the thymus, precursor cells undergo a complex set of developmental events and emerge as mature T lymphocytes capable of specific antigen recognition. We have found that there is a key developmental phase during the double negative stage of thymic development in which the cells resemble antigen-activated mature cells. We have made preparations of day 14/15 triple negative fetal thymocytes that exhibit the activated T lymphocyte markers CD25, ICAM-1, Ly-6A/E, CD44, and HSA and are rapidly proliferating as evidenced by flow cytometric examination of BrdU incorporation. We found that the gene regulators NF-kappaB, the NF-kappaB p50 homodimer complex, NF-AT, oct-1, oct-2, AP-1, and SRF, are all were active in these early thymocytes. Whereas the octamer factors and SRF persist during ontogeny, NF-kappaB, NF-AT, and AP-l decrease and are undetectable in the adult thymus. Thus, transcriptional events that are usually triggered by antigen stimulation in mature T cells take place early in thymic ontogeny in the absence of the T cell receptor. Our analysis suggests that there are striking regulatory similarities but also important differences between the activation processes that take place in antigen-stimulated mature T cells and thymic progenitor cells. We are now developing an in vitro system that recapitulates early steps in T cell differentiation to determine the molecular signals required. As part of this effort we are beginning to produce targeted mutations in signalling molecule genes to determine their effect on thymic development.